DESCRIPTION: The coupling between cell surface receptors and intracellular effectors is mediated by a family of G proteins composed of a complex of G alpha and G beta/gamma subunits. The activation of G proteins involves a receptor-catalyzed nucleotide exchange reaction that generates free G-alpha/GTP and G-beta/gamma leading to activation of the effector enzymes. Phosducin is a phosphoprotein that forms a specific complex with G-beta/gamma and inhibits the G-protein coupled cascade. The goal of the proposed research is to elucidate the molecular interaction of phosducin and G-beta/gamma. Three different approaches will be taken. (1) The solution dynamics of the phosducin/G-beta/gamma complex will be studied with the aid of fluorescence spectroscopy. A fluorescent probe will be used to label G-beta/gamma to monitor the formation and dissociation of the complex. The affinity of various G- beta/gamma subunits for phosducin and the stability of the complex with respect to the phosphorylation of phosducin will be evaluated. (2) The molecular structure of the phosducin/G-beta/gamma complex will be determined by X-ray crystallography. Single crystals of the phosducin and transducin G- beta/gamma subunit complex from retinal photoreceptor cells have been obtained. A major effort will be devoted to solving the three-dimensional structure of the complex. Attempts will be made to obtain similar crystals of phosducin complexed with other G-beta/gamma subunits. (3) The well-developed molecular genetics of the yeast Saccharomyces cerevisiae will be exploited to study regulation of G- beta/gamma in vivo. The phosducin gene will be introduced into yeast to monitor the effects of mutations on the yeast mating response, which is stimulated by G-beta/gamma. The interaction between phosducin and T-beta will be analyzed genetically using the two-hybrid system in yeast. Efforts will be made to identify a homologue of phosducin in yeast in order to study modulation of G-beta/gamma activity in a native signaling system amenable to genetic analysis. Integrating the results obtained from biochemical characterization, X-ray diffraction and the molecular genetic study, will facilitate determination of the interaction among phosducin, G-beta and G-gamma and the regulation of the phosducin/G- beta/gamma complex. Understanding the structural constraints of the interaction between phosducin and G-beta/gamma may lead to novel approaches in the design of drugs for the treatment of diseases involving G-protein coupling action.